New supporting data to guide the use of evident toxicity in acute oral toxicity studies (OECD TG 420).

Currently there are three test guidelines (TG) for acute oral toxicity studies of substances or mixtures from the Organisation for Economic Co-operation and Development (OECD). TG 423 and TG 425 use lethality as an endpoint, while TG 420 replaces death with 'evident toxicity', defined as clear signs that exposure to a higher dose would result in death. However, the perceived subjectivity of 'evident toxicity' may be preventing wider use of TG 420. To address this, the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) and the European Partnership for Alternative Approaches to Animal Testing (EPAA) collaborated to provide recommendations on the recognition of 'evident toxicity'. Historical data from acute oral toxicity studies were analysed for clinical signs at the lower dose that could have predicted death at the higher dose. Several signs including ataxia, laboured respiration, and eyes partially closed, alone or in combination, are highly predictive. Others such as lethargy, decreased respiration, and loose faeces have lower but still appreciable positive predictive value (PPV). The data has been used to develop recommendations to promote use of TG 420 and thus reduce the suffering and numbers of animals used in acute oral toxicity studies.

Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals.

Animal testing has long been integral to the development of biologicals, including vaccines. The use of animals can provide important information on potential toxicity, insights into their mechanism of action, pharmacokinetics and dynamics, physiologic distribution, and potency. However, the use of these same methods is often adopted into the post-licensure phase of the product life cycle for the monitoring of product qualities, such as potency or safety, as part of their routine batch release. The UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) and the World Health Organization (WHO) are collaborating on a project to review animal-based testing methods described in WHO manuals, guidelines and recommendations for biologicals to identify where updates can lead to a more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction, and Refinement of animal tests) in batch release testing requirements. An international working group consisting of more than 30 representatives from pharmaceutical and biotechnology companies, national control laboratories and regulatory bodies is performing this review. This project aims to address concerns about inconsistencies in the guidance for the scientifically justified use of animal methods required for the post-licensure quality control and batch release testing of biologicals, and the near absence of recommendations for the application of 3Rs principles within the relevant guidelines. Improved adoption of 3Rs principles and non-animal testing strategies will help to reduce the delays and costs associated with product release testing and help support faster access to products by the global communities who need them most urgently.

Preclinical screening for antidepressant activity - shifting focus away from the Forced Swim Test to the use of translational biomarkers.

Depression is the world's predominant mental health problem and a leading cause of disability. Neuropharmacological research has not yet advanced treatments to sufficiently meet clinical need, largely due to the failure of animal models to predict clinical efficacy. The forced swim test (FST) has been extensively used in the field of antidepressant research but has been under scrutiny due to its perceived severity to animals. Any use of animals in experiments and testing must have a scientific or regulatory purpose and researchers need to ensure that there is no scientifically valid alternative. However, regulatory requirements have been incorrectly cited as a reason to support the use of the FST. More research is required on tests that do not involve stressing animals as replacements for the FST. Non-behavioural neurochemical measures might provide a means to advance neuropharmacological developments while reducing animal suffering. For example, brain-derived neurotrophic factor (BDNF) may be promising.

An evaluation of the fixed concentration procedure for assessment of acute inhalation toxicity.

Acute inhalation studies are conducted in animals as part of chemical hazard identification and for classification and labelling. Current methods employ death as an endpoint (Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 403 and TG436) while the recently approved fixed concentration procedure (FCP) (OECD TG433) uses fewer animals and replaces lethality as an endpoint with evident toxicity. Evident toxicity is the presence of clinical signs that predict that exposure to the next highest concentration will cause severe toxicity or death in most animals. Approval of TG433 was the result of an international initiative, led by the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs), which collected data from six laboratories on clinical signs recorded for inhalation studies on 172 substances. This paper summarises previously published data and describes the additional analyses of the dataset that were essential for approval of the TG.

Drivers and barriers in the consistency approach for vaccine batch release testing: Report of an international workshop.

Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine, and the principle that the quality of subsequent batches is the consequence of the application of consistent production of batches monitored by a GMP quality system. Efforts to implement the consistency approach are supported by several drivers from industry, government, and research, but there are also several barriers that must be overcome. A workshop entitled "Consistency Approach, Drivers and Barriers" was organized, which aimed to discuss and identify drivers and barriers for the implementation of the 3Rs in the consistency approach from three different perspectives/domains (industry, regulatory and science frameworks). The workshop contributed to a better understanding of these drivers and barriers and resulted in recommendations to improve the overall regulatory processes for the consistency approach. With this report, we summarise the outcome of this workshop and intend to offer a constructive contribution to the international discussion on regulatory acceptance of the consistency approach.

Steps towards the international regulatory acceptance of non-animal methodology in safety assessment.

The current animal-based paradigm for safety assessment must change. In September 2016, the UK National Centre for Replacement, Refinement and Reduction of Animals in Research (NC3Rs) brought together scientists from regulatory authorities, academia and industry to review progress in bringing new methodology into regulatory use, and to identify ways to expedite progress. Progress has been slow. Science is advancing to make this possible but changes are necessary. The new paradigm should allow new methodology to be adopted once it is developed rather than being based on a fixed set of studies. Regulatory authorities can help by developing Performance-Based Standards. The most pressing need is in repeat dose toxicology, although setting standards will be more complex than in areas such as sensitization. Performance standards should be aimed directly at human safety, not at reproducing the results of animal studies. Regulatory authorities can also aid progress towards the acceptance of non-animal based methodology by promoting "safe-haven" trials where traditional and new methodology data can be submitted in parallel to build up experience in the new methods. Industry can play its part in the acceptance of new methodology, by contributing to the setting of performance standards and by actively contributing to "safe-haven" trials.

Applying the 3Rs to non-human primate research: Barriers and solutions.

Progress is being made in the development and application of methods to replace, reduce and refine the use of non-human primates (NHPs) in biomedical research and testing of products and devices. However, there remain considerable cultural and practical barriers to widespread uptake of available 3Rs techniques and to further advancement of the 3Rs in NHP research, over and above scientific obstacles. While most of these barriers apply also to the use of other vertebrate species, there is arguably a greater imperative to overcome them in the case of the NHPs, given their high sentience and the degree of societal concern about their use. To do so will require greater awareness among researchers of the availability and scientific benefits of 3Rs approaches; increased funding for the development of new research models and tools, infrastructure and training; more robust scientific and ethical review of research proposals involving NHPs; better retrospective evaluation of the benefits accrued from NHP research; and improved knowledge transfer. Change is not made without inconvenience, but fully applying the 3Rs to research involving NHPs can improve the quality of science, its translation, business efficiency and public support.

A global initiative to refine acute inhalation studies through the use of 'evident toxicity' as an endpoint: Towards adoption of the fixed concentration procedure.

Acute inhalation studies are conducted in animals as part of chemical hazard identification and characterisation, including for classification and labelling purposes. Current accepted methods use death as an endpoint (OECD TG403 and TG436), whereas the fixed concentration procedure (FCP) (draft OECD TG433) uses fewer animals and replaces lethality as an endpoint with 'evident toxicity.' Evident toxicity is defined as clear signs of toxicity that predict exposure to the next highest concentration will cause severe toxicity or death in most animals. A global initiative including 20 organisations, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) has shared data on the clinical signs recorded during acute inhalation studies for 172 substances (primarily dusts or mists) with the aim of making evident toxicity more objective and transferable between laboratories. Pairs of studies (5 male or 5 female rats) with at least a two-fold change in concentration were analysed to determine if there are any signs at the lower dose that could have predicted severe toxicity or death at the higher concentration. The results show that signs such as body weight loss (>10% pre-dosing weight), irregular respiration, tremors and hypoactivity, seen at least once in at least one animal after the day of dosing are highly predictive (positive predictive value > 90%) of severe toxicity or death at the next highest concentration. The working group has used these data to propose changes to TG433 that incorporate a clear indication of the clinical signs that define evident toxicity.

Advancing the 3Rs in regulatory toxicology - Carcinogenicity testing: Scope for harmonisation and advancing the 3Rs in regulated sectors of the European Union.

Different government agencies operating in the European Union regulate different types of chemical products but all require testing for carcinogenicity to support applications for product marketing and commercialisation. A conference was held in Brussels in 2013 where representatives of the pharmaceutical, animal health, chemical and plant protection industries, together with representatives of regulatory agencies, universities and other stakeholders, met under the auspices of The European Partnership for Alternative Approaches to Animal Testing (EPAA) to discuss the varying requirements for carcinogenicity testing, and how these studies might be refined to improve hazard evaluation and risk assessment while implementing principles of the 3Rs (replacement, refinement and reduction in animal studies). While there are some similarities, the regulatory approaches in pharmaceutical, animal health, chemical and plant protection sectors have varying degrees of flexibility in requirements for carcinogenicity testing, to an extent reflecting concerns over the magnitude and duration of human exposure, either directly as in therapeutic exposure to pharmaceuticals, or indirectly through the ingestion of residues of veterinary drugs or plant protection chemicals. The article discusses these differences and other considerations for modified carcinogenicity testing paradigms on the basis of scientific and 3Rs approaches.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Improving Translational Relevance in Preclinical Psychopharmacology (iTRIPP).

Animal models are important in preclinical psychopharmacology to study mechanisms and potential treatments for psychiatric disorders. A working group of 14 volunteers, comprising an international team of researchers from academia and industry, convened in 2021 to discuss how to improve the translational relevance and interpretation of findings from animal models that are used in preclinical psychopharmacology. The following paper distils the outcomes of the working group's discussions into 10 key considerations for the planning and reporting of behavioural studies in animal models relevant to psychiatric disorders. These form the iTRIPP guidelines (Improving Translational Relevance In Preclinical Psychopharmacology). These guidelines reflect the key considerations that the group thinks will likely have substantial impact in terms of improving the translational relevance of behavioural studies in animal models that are used to study psychiatric disorders and their treatment. They are relevant to the research community when drafting and reviewing manuscripts, presentations and grant applications. The iTRIPP guidelines are intended to complement general recommendations for planning and reporting animal studies that have been published elsewhere, by enabling researchers to fully consider the most appropriate animal model for the research purpose and to interpret their findings appropriately. This in turn will increase the clinical benefit of such research and is therefore important not only for the scientific community but also for patients and the lay public.

Consensus document on European brain research.

Psychiatric and neurological diseases combined represent a considerable social and economic burden in Europe. A recent study conducted by the European Brain Council (EBC) quantified the 'cost and burden' of major brain diseases in Europe, amounting to €386bn per year. Considering that these costs will increase exponentially in the years to come due to ageing of the European population, it is necessary to act now in order to curb this increase and possibly reverse the trend. Thus, establishing a strong European platform supporting basic and clinical research in neuroscience is needed to confront the economic and social challenge posed by management of brain diseases in European countries. To setup a platform for discussion, EBC published in 2006 a Consensus Document on European Brain Research, describing needs and achievements of research in Europe and presenting proposals for future research programs. Since 2006, European research in neuroscience has advanced tremendously. The present document represents an update elaborated to reflect changes in research priorities and advances in brain research that have taken place since 2006. The same approach and format have been used here as in the previous version. Multinational and multidisciplinary teams have once again come together to express their views, not only on the current strengths in European research, but also on what needs to be done in priority, hoping that this update will inspire policy makers and stakeholders in directing funding for research in Europe.

Preclinical safety testing of monoclonal antibodies: the significance of species relevance.

Selecting a pharmacologically relevant animal species for testing the safety and toxicity of novel monoclonal antibody (mAb) therapies to support clinical testing can be challenging. Frequently, the species of choice is the primate. With the increased number of mAbs in the pharmaceutical pipeline, this has significant implications for primate use, and so raises several important scientific, ethical and economic issues. Here, following a recent international workshop held to debate this topic, we discuss issues in the preclinical testing of mAbs, with a particular focus on species relevance and primate use, and provide suggestions for how these issues might be addressed.

Consensus document on European brain research.

Brain disease psychiatric and neurologic disease combined represents a considerable social and economic burden in Europe. Data collected by the World Health Organization (WHO) suggest that brain diseases are responsible for 35% of Europe's total disease burden. An analysis of all health economic studies of brain diseases in Europe, published by the European Brain Council (EBC) in June 2005, estimated the total cost of brain disease in Europe in 2004 to be Euro 386 billion. That burden is set to grow, mainly due to the fact that the European population is ageing. Investment in brain sciences does not match that burden now, let alone in the future. Brain research received only 8% of the life science budget in the European Commission's Fifth Framework Programme, which represents less than 0.01% of the annual cost of brain disorders for that period. Over the last decade, Europe has been losing ground to the USA and Japan in terms of both basic and clinical research. Many of Europe's young researchers are taking up posts in the USA and staying there. Big pharmaceutical companies are fleeing Europe for the USA, taking their drug development programmes with them. Research in the brain sciences now holds the promise of therapies that halt and even reverse neurodegeneration, of better diagnostic tools, neural prostheses for the paralysed and drugs for depression and anxiety that are tailored to the individual, thereby eliminating or reducing side effects. Our growing understanding of the normal brain could lead to better prevention of brain disease and to more effective teaching methods. The need for innovative treatments has never been greater, and Europe boasts clusters of excellent researchers in biotechnology who could collaborate with brain scientists and the pharmaceutical industry to realise this promise. But if Europe is to seize these opportunities and meet the challenge of brain disease, it needs to go forward on the basis of greater collaboration between countries, greater collaboration between industry, academia and patient organisations, and increased investment in the brain sciences. The EBC was formed in 2002 to bring together scientists, clinicians, the pharmaceutical industry, charities and patient organisations from all over Europe to campaign for these goals. It takes a novel, bottom-up approach to research policy, and in developing this consensus document, it aims to promote a greater and more focused effort in this area, to improve public understanding of the brain sciences and above all, to support brain research as a priority under the European Commission's Seventh Framework Programme (FP7, 2007-2013). The research programme outlined here was first conceived by the EBC board. An outline was sent to all member organisations and a number of individual experts for comments. Following that, a table of contents was developed. The 45 research themes were written by groups of experts from across Europe who represent a wide range of disciplines. Each one contains a proposal for future research on a specific brain-related theme which the EBC believes could form the basis of one or more integrated projects or strategic targeted research projects (STREP) funded under FP7. The EBC has deliberately focused on the major diseases and then described the basic research needed to understand and treat or perhaps even cure those diseases. The programme is therefore constructed "from man to molecule" and not the other way round, with equal importance attached to basic and clinical research. The EBC suggests that each of the proposed integrated projects or STREP should be awarded a budget in the order of Euro 10 to 15 million. In addition, brain research should be treated as an important element of many other parts of FP7, such as the European Research Council and research programmes on information technology and the causes of violence. Any research programme that concerns human behaviour should, by definition, take account of brain research. The EBC envisages that the priority for brain research it proposes at the European level will translate into higher priority for brain research at the national level, and this document may also serve as a starting point for the development of national consensus programmes. It seems likely that consensus conferences on brain research in Europe may further develop the themes and ideas discussed here. An EBC task force may also be established to further the consensus process. In general, increasing funding in the brain sciences would bring enormous economic returns by lightening the burden on healthcare systems and increasing the productivity of affected individuals-and might easily pay for itself. The human and social returns of such an investment are inestimable. And the time to act is now.

Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges.

Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio™, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory. We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors have made recommendations for a data-driven approach to the toxicological assessment of mAb biosimilars that minimises unnecessary use of animals and can be used across all regions of the world.

Opportunities for improving animal welfare in rodent models of epilepsy and seizures.

Animal models of epilepsy and seizures, mostly involving mice and rats, are used to understand the pathophysiology of the different forms of epilepsy and their comorbidities, to identify biomarkers, and to discover new antiepileptic drugs and treatments for comorbidities. Such models represent an important area for application of the 3Rs (replacement, reduction and refinement of animal use). This report provides background information and recommendations aimed at minimising pain, suffering and distress in rodent models of epilepsy and seizures in order to improve animal welfare and optimise the quality of studies in this area. The report includes practical guidance on principles of choosing a model, induction procedures, in vivo recordings, perioperative care, welfare assessment, humane endpoints, social housing, environmental enrichment, reporting of studies and data sharing. In addition, some model-specific welfare considerations are discussed, and data gaps and areas for further research are identified. The guidance is based upon a systematic review of the scientific literature, survey of the international epilepsy research community, consultation with veterinarians and animal care and welfare officers, and the expert opinion and practical experience of the members of a Working Group convened by the United Kingdom's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs).

7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine: a functionally selective gamma-aminobutyric acid(A) (GABA(A)) alpha2/alpha3-subtype selective agonist that exhibits potent anxiolytic activity but is not sedating in animal models.

There is increasing evidence that compounds with selectivity for gamma-aminobutyric acid(A) (GABA(A)) alpha2- and/or alpha3-subtypes may retain the desirable anxiolytic activity of nonselective benzodiazepines but possess an improved side effect profile. Herein we describe a novel series of GABA(A) alpha2/alpha3 subtype-selective agonists leading to the identification of the development candidate 17, a nonsedating anxiolytic in preclinical animal assays.

The future of non-human primate use in mAb development.

It has been predicted that the use of non-human primates (NHPs) is going to increase considerably in the development of monoclonal antibodies (mAbs). Opportunities exist to focus on a rigorous, science-based approach to drug development, however, which will minimize this increase. In this article, the authors review current and future NHP use in mAb development based on surveys, experience and expert opinion and propose a framework that will minimize future NHP use and continue to support science and innovation.

Preclinical development of monoclonal antibodies: considerations for the use of non-human primates.

The development of mAbs remains high on the therapeutic agenda for the majority of pharmaceutical and biotechnology companies. Often, the only relevant species for preclinical safety assessment of mAbs are non-human primates (NHPs), and this raises important scientific, ethical and economic issues. To investigate evidence-based opportunities to minimize the use of NHPs, an expert working group with representatives from leading pharmaceutical and biotechnology companies, contract research organizations and institutes from Europe and the USA, has shared and analyzed data on mAbs for a range of therapeutic areas. This information has been applied to hypothetical examples to recommend scientifically appropriate development pathways and study designs for a variety of potential mAbs. The addendum of ICHS6 provides a timely opportunity for the scientific and regulatory community to embrace strategies which minimize primate use and increase efficiency of mAb development.

TPA023 [7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine], an agonist selective for alpha2- and alpha3-containing GABAA receptors, is a nonsedating anxiolytic in rodents and primates.

7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) is a triazolopyridazine that binds with equivalent high (subnanomolar) affinity to the benzodiazepine binding site of recombinant human GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has partial agonist efficacy at the alpha2 and alpha3 subtypes and essentially antagonist efficacy at the alpha1 and alpha5 subtypes. In rats, TPA023 gave time- and dose-dependent occupancy after oral dosing, with 50% occupancy corresponding to a dose of 0.42 mg/kg. It has anxiolytic-like activity in unconditioned (elevated plus maze) and conditioned (fear-potentiated startle and conditioned suppression of drinking) rat models of anxiety with minimum effective doses (MED; 1-3 mg/kg) corresponding to 70 to 88% occupancy. However, there was no appreciable sedation in a response sensitivity (chain-pulling) assay at a dose of 30 mg/kg, resulting in 99% occupancy. Similarly, TPA023 was robustly anxiolytic in the squirrel monkey conditioned emotional response assay, with a MED of 0.3 mg/kg, but did not produce any sedation in a lever-pressing test of sedation even at 10 mg/kg. TPA023 produced no impairment in performance in the mouse Rotarod assay, and there was only a mild interaction with ethanol. In addition to anxiolytic-like efficacy, TPA023 had anticonvulsant activity in a mouse pentylenetetrazole seizure model. Finally, TPA023 did not cause precipitated withdrawal in mice treated for 7 days with the nonselective agonist triazolam, nor did N-methyl-beta-carboline-3-carboxamide (FG 7142) precipitate withdrawal in mice treated for 7 days with TPA023. In summary, the novel alpha2/alpha3-selective efficacy profile of TPA023 translates into a nonsedating anxiolytic profile that is distinct from nonselective agonists.